The Molecular Mechanisms Of H₂S Exposure-Induced Broiler Spleen Necroptosis Based On MAPKs/NF-?B Pathway

Hydrogen sulfide?H₂S?is one of the main pollutants in the atmosphere.In recent years,with the booming development of livestock and poultry industry,a large amount of H₂S was produced in the decomposition process of livestock and poultry excreta.If ventilation is poor,excessive H₂S accumulated in livestock and poultry houses will seriously damage the health of livestock and poultry.Studies have shown that the exposure of H₂S in chicken coop reduces the production performance,meat quality and immune function of chickens,and further studies have found that excessive H₂S exposure leads to oxidative stress,inflammatory response,energy metabolism dysfunction and other damage characteristics in multi-tissue organs of broilers.Necroptosis is a new form of cell death discovered in recent years and is one of the important manifestations of tissue and organ damage induced by environmental exposure.In order to explore the molecular mechanisms of spleen necroptosis caused by excessive H₂S exposure in broiler chickens and the signaling pathway involved,120 1-day-old Ross 308 male chickens were randomly assigned to two airless chicken coops equipped with automatic gas monitoring equipment.No H₂S gas was injected in the control chicken coop?H₂S < 2.0 mg/m3?.H₂S gas was injected into the chicken coop of the H₂S group,and the H₂S concentration was kept at 4.0 ± 0.5 mg/m3?0-3 weeks?and 20.0 ± 0.5 mg/m3?4-6 weeks?.Routine feeding and free intake of food and water were conducted for two groups of broilers.At 2 weeks,4 weeks and 6 weeks,10 broilers were randomly selected from each group for painless death,and spleen tissues were collected.In this study,the observation of chicken spleens histopathological and ultrastructural changes,the detection of m RNA and protein expressions of necroptosis,energy metabolism,MAPKs pathway,NF-?B pathway,inflammatory factors,and antioxidant index such as GSH,CAT,T-AOC,SOD were carried out.The results showed that:?1?The pathological structure of the spleens of broilers in the H₂S group had obvious changes,including spleen damage characteristics,the number of lymphocytes and macrophages in the spleen tissue of H₂S group was significantly reduced.Nuclear rupture,nuclear lysis and erythrocythemia also appeared in the H₂S group spleens.The results of ultrastructural observation showed typical cell necrosis characteristics including mitochondrial swelling or even vacuolation,cell membrane rupture and dissolution,cytosolic content spillover,and accompanied with extensive vesicular formation.?2?Compared with the corresponding control group,the m RNA expressions of RIPK1,RIPK3,TAK1,TAB2 and TAB3 in the H₂S group were significantly increased,while the m RNA expression of Caspase-8 was significantly decreased.Similarly,the protein expressions of RIPK1 and RIPK3 in the H₂S group were significantly higher than those in the corresponding control group,while the protein expression of Caspase-8 was significantly decreased.In addition,the phosphorylated MLKL expression was significantly increased.The GSH content,CAT,T-AOC and SOD activities were significantly decreased in the H₂S-exposed spleens,while the i NOS activity,NO,H₂O₂ and MDA contents were significantly increased.The activities of four ATPases were decreased,and other energy metabolism-related genes except NOX2 were significantly reduced.?3?With the extension of H₂S exposure time,MAPKs pathway related expressions were increased in a time-dependent manner,shows that excessive H₂S gas exposure can activate MAPKs pathway.?4?Further detection of phosphorylated protein expressions of NF-?B pathway-related genes?IKK?/?,NF-?B p65,I?B??,the m RNA and protein expressions of inflammatory response-related genes?TNF-?,IL-1?,HSP70 and HSP90?.The results showed that exposure to excessive H₂S gas activated NF-?B pathway and resulted in inflammatory response in broilers spleens.